Mucin 1 (MUC1) is aberrantly overexpressed in ~90% of human breast cancers and, as such, has been regarded as a highly attractive target for the development of new anti-cancer agents. However, progress in the identification of drugs that block MUC1 had been previously limited by a lack of understanding as to how MUC1 contributes to malignant progression. In this regard, MUC1 consists of two subunits, and early research focused on the shed extracellular mucin subunit (MUC1-N). Work supported by this grant has now demonstrated that the transmembrane MUC1-C subunit functions as an oncoprotein and is a druggable target for the development of novel therapeutic agents. MUC1-C represents a potentially selective target in that the Muc1 knock-out mouse is viable and has no evident phenotype. In addition, the MUC1-C subunit is normally positioned in an inactive state at the apical borders of mammary epithelial cells. With transformation and loss of polarity, MUC1-C associates with receptor tyrosine kinases at the breast cancer cell membrane and localizes to the cytoplasm, nucleus and mitochondria. The potential selectivity of MUC1-C as a target is further supported by the demonstration that MUC1-C inhibitors are highly effective in the treatment of human breast tumor xenografts in mice without normal tissue toxicity. The overall objective of the proposed work is to improve scientific knowledge, therapeutic capability and clinical practice by targeting MUC1-C with novel approaches that block its oncogenic function. Our hypothesis is that the MUC1-C oncoprotein is a druggable target that can be inhibited at the cell membrane and within the breast cancer cell. The proposed work will address this hypothesis by providing (i) new insights into how inhibition of MUC1-C function blocks growth and survival of breast cancer cells, and (ii) new therapeutic agents that will be evaluated preclinically for potential development in the clinic for the treatment of breast cancer. The Specific Aims are: (1) To assess potential therapeutic approaches that target the MUC1-C subunit extracellular domain at the breast cancer cell surface; (2) To evaluate the development of orally bioavailable MUC1-C peptide inhibitors that block its function in the cytoplasm; (3) To define the activity of peptide and small molecule inhibitors of MUC1-C function in the nucleus and mitochondria; and (4) To identify effective combinations of MUC1-C inhibitors with targeted anti-cancer agents for breast cancer treatment.